Project Summary Dry eye disease (DE) is a multifactorial condition defined by signs of tear film instability and symptoms of ocular irritation and blurred vision. Symptom relief is the primary reason DE patients seek medical attention. However, topical eye treatments that reduce the signs of ocular inflammation often fail to manage symptoms in moderate to severe cases and peripheral signs of DE do not reliably predict disease severity. These findings suggest a critical role for CNS mechanisms in the development and maintenance of ocular pain in severe cases of DE; however, little is known about central neural processing of ocular signals that are relevant for symptomatic DE. Converging lines of evidence from electrophysiological, molecular and anatomical approaches are applied in a rodent model for tear deficiency, exorbital gland removal. The overall goals of this project are to determine mechanisms for increased neural excitability of trigeminal brainstem neurons after persistent tear reduction and if neuron- glia interactions contribute to altered neural processing and enhanced protective eye muscle reflexes. Aim 1 develops a quantitative sensory testing (QST) protocol to characterize the encoding properties of ocular trigeminal brainstem neurons in male and female rats. A novel recording and analysis method is developed to assess squint-like activity in upper and lower portions of the orbicularis oculi muscle as a measure of nociceptive behavior in anesthestized rats. Aim 2 determines if a ?feed forward? pathway connecting caudal with rostral trigeminal regions contributes to ocular hyperalgesia in DE. Aim 3 determines: a) if sensory signals that drive microglia activation do so by upregulating Toll-like and purinergic P2x receptors, b) the distribution and localization neuroactive products by microglia (IL-1?, BDNF) and their receptors on neurons and glia, and c) blockade of neuron-glia interactions alter the properties of ocular-responsive neurons and evoked eyelid muscle responses in sham and DE male and female rats. By combining neural recording and eye muscle activity with approaches that interfere with microglia activation (purinergic receptors), inflammasome formation (NLRP3) and products of microglia (IL-1?), this project will provide novel information on central mechanisms of ocular hypersensitivity in an animal model for tear deficiency. The long-term goals of this project are to develop new approaches to assess symptoms in moderate to severe cases of DE and to determine if targeting receptors for neuron- microglia interactions has therapeutic potential to manage ocular hyperalgesia in DE.